Many patients with pulmonary diseases, such as acute respiratory distress syndrome and emphysema, require hyperoxia to maintain adequate tissue oxygenation. However, hyperoxia although beneficial can further perpetuate/exacerbate acute lung injury (ALI). Through a genetic linkage analysis, we have first identified Nrf2 transcription factor as an important candidate gene that regulates hyperoxic lung injury (HLI). The overall objective of the previous proposal was to examine the role of Nrf2 and its downstream target genes involved in HLI. During the last funding period, we have shown that Nrf2-deficient (Nrf2-/-) mice have a greater susceptibility to hyperoxia induced lung injury and inflammatory responses, compared with wildtype (Nrf2+/+) mice. This was primarily due to a diminished lower level of both basal and inducible expression of antioxidant enzymes (AOEs), which are critical to detoxify reactive oxygen (ROS) and/or nitrogen (RNS) species, in Nrf2-/- mice. Genetic variations appear to play a significant role in modulating the development of complex disorders such as ALI. Since NRF2 is a master regulator of cellular oxidative stress, a major determinant that can contribute to ALI, we have analyzed polymorphisms in the coding and non-coding regions of NRF2 gene. To this end, we found novel functional single nucleotide polymorphisms (SNPs) in the promoter region of NRF2 that were associated with at-risk patients with major trauma. Thus, our preliminary findings provide an important framework for expanding the scope of this project to determine the functional relevance of NRF2 promoter polymorphisms. We hypothesize that perturbation either in specific signaling or factors controlling the NRF2 expression and activation may result in lower levels of antioxidant enzyme expression, thereby contributing to or enhancing susceptibility to the development of respiratory pathogenesis. To test our hypothesis, we propose the following four Specific Aims: 1) To determine the functional importance of novel SNPs in regulation of NRF2 transcription. 2) To Identify the trans-factor(s) that modulate NRF2 transcription and determine whether or not the binding of these transcriptional factors to the NRF2 promoter is altered by hyperoxia and relevant to the SNPs. 3) Elucidate the signal transduction pathways that lead to NRF2 dissociation from the NRF2:KEAP1 complex in response to acute and chronic hyperoxia exposure in vitro. 4) Investigate whether PI3K-Akt signaling via Nrf2 regulates HLI and whether boosting antioxidant gene expression by activating Nrf2 using a synthetic triterpenoid, which work at very low doses, will confer protection against HLI in mice. Our overall findings may have important implications to identify the effector mechanisms causing susceptibility to ALI and may be helpful to modulate the development of this complex disorder and other respiratory diseases that have been linked to oxidant stress. In addition, the results of this study will provide a strong rationale to further explore the use of this triterpenoid as potential therapeutic agents in modulating HLI. NRF2-dependent transcriptional response is vital to mitigate cellular stress induced by various oxidants and toxicants, our overall findings may have important implications to identify the effector mechanisms causing susceptibility to acute lung injury and may be helpful to modulate development of this complex disorder and other respiratory diseases that have been linked to oxidant stress.